Methods and compositions high scale therapeutic production of memory nk cells

ABSTRACT

Disclosed are compositions and methods relating to the expansion of memory NK cells.

I. BACKGROUND

1. Hematopoietic stem cell transplantation (HSCT) from genotypicallyHLA-matched siblings has improved long-term survival in patients withhematologic cancer malignancies and marrow failure syndromes. Everyyear, more than 10,000 Americans get life-threatening diseases for whichthe only hope of a cure is a bone marrow transplant from an unrelateddonor or cord blood unit. However, more than 70% of patients who couldbenefit from an allogeneic stem cell transplant do not have a matchedsibling donor. These circumstances delay treatment, making it necessaryto resort to less than optimal use of a partially mismatched donor,which eventually leads to increased incidence of graft-versus-hostdisease (GVHD), graft failure, and relapse, all of which dramaticallydecrease patient survival.

2. Additional limitations are posed by the duration and the costlyfinancial, mental, and health burdens of the transplant process. Thus,the application of HSCT from an unrelated donor is limited to younger,healthier patients with appropriate socioeconomic support that canendure the process. Further challenges are posed by the high rate ofrelapse due to the inability to eradicate residual cancer cells.Although HSCT is considered to be curative, cancer relapse rates arestaggering. Thus, novel, more targeted immunotherapies are needed thatwould be more effective, preferably without the need for a matcheddonor. Donor lymphocyte infusion (DLI), for the treatment of acutemyeloid leukemia (AML) relapse after HSCT was introduced in 1990s. Thisapproach consisted of the administration of lymphocytes from theoriginal donor to the AML patient with relapsed disease. Yet, clinicalbenefits were limited and observed only in a minority of patients withsmaller tumor burdens, and T cell mediated GVHD often further worsenedthe outcomes.

3. A significant portion of donor lymphocyte infusion mediatedgraft-versus-tumor (GVT) effect may be due to natural killer (NK) cells.The infusion of NK cells isolated from donor blood could producebeneficial GVT effects without causing GVHD. Preclinical and clinicaldata has shown effectiveness of NK cell infusions leading towardcomplete remission without any GVHD. Thus, NK cell infusion, incombination with autologous transplantation, or as a standalonetreatment, offers an innovative, and potentially very effective,alternative for those patients who do not have a matched donor,experience relapse, or do not qualify for transplant. However, these NKcell treatments create demand for NK cell expansion sufficient in numberto provide therapeutic treatment. Accordingly, there is a great need fornew and improved methodologies aimed at increasing memory NK cellnumbers.

4. Additional advantages of the disclosed method and compositions willbe set forth in part in the description which follows, and in part willbe understood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

II. SUMMARY

5. The present application generally relates to compositions and methodscomprising memory natural killer (NK) cells. More particularly, theapplication relates to the in vivo, ex vivo, or in vitro stimulation andexpansion of memory natural killer (NK) cells, which are capable ofattacking and killing cancer cells, virally infected cells and certainimmune cells.

6. Disclosed herein are methods for increasing the number of memory NKcells comprising a) preactivating NK cells by contacting at least one NKcell with at least one or more stimulatory cytokines; b) expanding thepreactivated NK cells of step a) by contacting said cells with a vesiclecomprising an NK cell effector agent (for example, contacting with PM21particles, EX21 exosomes, or FC21 feeder cells). In some aspect aremethods for increasing the number of memory NK cells further comprisingwashing after the preactivation step a) and/or the expansion step b)and/or resting the memory NK cells cells after the expansion step b).

7. In one aspect disclosed herein are methods for increasing the numberof memory NK cells wherein the NK cells for use in the disclosed methodsare obtained from an unselected population of peripheral bloodmononuclear cells.

8. Also disclosed are methods of any preceding aspect wherein the atleast one or more stimulatory cytokines are selected from the groupconsisting of IL-12, IL-15, and IL-18. In one aspect, the methods cancomprise contacting the NK cells with 3 stimulatory cytokines.

9. Also disclosed are methods of any preceding aspect wherein the NKcells are contacted with the IL-12, IL-15, or IL-18 in vitro, in vivo,or ex vivo.

10. Also disclosed are methods of any preceding aspect wherein the NKcells are contacted with the one or more stimulatory cytokines for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 36, 48, 60, or 72 hours.

11. Also disclosed are methods of any preceding aspect furthercomprising contacting the NK cell with a cytokine selected from thegroup consisting of 4-1BBL, IL-2, IL-21, MICA/B, ULBP2, ICAM-1, 2B4,BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

12. In one aspect, disclosed herein are methods wherein the PM21particles, EX21 exosomes, or FC21 feeder cells comprise one or morestimulatory peptides coupled to a membrane-inserting peptide.

13. Also disclosed are methods of any preceding aspect whereinmembrane-inserting cytokine comprises a fused peptide that is capable ofmembrane insertion, with affinity for a lipid bilayer, and wherein saidfused peptide comprises a segment of IG4, CD4, or a combination thereof.

14. Also disclosed are methods of any preceding aspect wherein themembrane-inserting peptide comprises human Fc, GPI, trans-membraneT-cell receptor, or pHLIP.

15. Also disclosed are methods of any preceding aspect wherein the oneor more stimulatory peptides are selected from the group consisting of4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA/B, ULBP2, ICAM-1, 2B4,BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

16. Also disclosed are methods of any preceding aspect wherein the oneor more stimulatory cytokines coupled to a membrane-inserting peptide isa fusion protein encoded by recombinant DNA.

17. Also disclosed are methods of any preceding aspect wherein the NKcells are contacted with the PM21 particles, EX21 exosomes, or FC21feeder cells in vitro, in vivo, or ex vivo.

18. Also disclosed are methods of any preceding aspect wherein the NKcells of step a) are contacted with PM21 particles, EX21 exosomes, orFC21 feeder cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 days.

19. In one aspect, disclosed herein are methods wherein the cells arememory NK cells rested for at least 1, 2, 3, 4, or 5 days.

20. In one aspect, disclosed herein are kits for increasing the numberof memory NK cells comprising one or more cytokine and one or morevesicle comprising an NK cell effector agent.

21. Also disclosed are kits of any preceding aspect, wherein the one ormore cytokines is selected from the group consisting of IL-12, IL-15,and IL-18.

22. Also disclosed are kits of any preceding aspect, wherein the one ormore vesicles comprising an NK cell effector agent is a PM21 particle,EX21 exosome, or FC21 feeder cell.

23. Also disclosed are kits of any preceding aspect, wherein the NK celleffector agent is selected from the group consisting of -1BBL, IL-2,IL-15, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112,CCR7, DAP12, and DAP10

III. BRIEF DESCRIPTION OF THE DRAWINGS

24. The accompanying drawings, which are incorporated in and constitutea part of this specification, illustrate several embodiments andtogether with the description illustrate the disclosed compositions andmethods.

25. FIG. 1 shows increased memory NK cell expansion following contactwith PM21 particles, K562-mb21 feeder cells (FC21 cells), andpreactivation with IL12, IL15, and IL18 followed by contact with PM21particles.

IV. DETAILED DESCRIPTION

26. Before the present compounds, compositions, articles, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods or specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting.

A. Definitions

27. As used in the specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apharmaceutical carrier” includes mixtures of two or more such carriers,and the like.

28. Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

29. In this specification and in the claims which follow, reference willbe made to a number of terms which shall be defined to have thefollowing meanings:

30. “Optional” or “optionally” means that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not.

31. The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds, compositions, articles, devices and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary and are not intended to limit the disclosure. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.), but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. or is at ambient temperature, and pressure is at or nearatmospheric.

32. Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

B. Method of Expanding Memory NK Cells

33. Human NK cells are a subset of peripheral blood lymphocytes definedby the expression of CD56 or CD16 and the absence of T cell receptor(CD3). NK cells sense and kill target cells that lack majorhistocompatibility complex (MHC)-class I molecules. NK cell activatingreceptors include, among others, the natural cytotoxicity receptors(NKp30, NKp44 and NKp46), and lectin-like receptors NKG2D and DNAM-1.Their ligands are expressed on stressed, transformed, or infected cellsbut not on normal cells, making normal cells resistant to NK cellkilling. NK cell activation is negatively regulated via inhibitoryreceptors, such as killer immunoglobin (Ig)-like receptors (KIRs),NKG2A/CD94, and leukocyte Ig-like receptor-1 (LIR-1). Engagement of oneinhibitory receptor may be sufficient to prevent target lysis. Hence NKcells efficiently target cells that express many stress-induced ligands,and few MHC class I ligands.

34. Infusions of NK cells are a treatment option for patients withcancers susceptible to NK cell lysis, including blood cancers (such asacute myeloid leukemia or multiple myeloma) and several solid tumors(e.g. brain tumor, Ewing sarcoma and rhabdomyosarcoma). Increasednumbers of functional NK cells can also significantly enhance theefficacy of therapeutic antibodies used in treatment of several cancers,including lymphomas, colorectal cancer, lung cancer, and breast cancer,among others. These types of personalized treatments are, however, verycostly, with a typical antibody-containing regimen costing tens ofthousands of dollars. Furthermore, the expected efficacy of existingmethods is often not achieved due to the lack of immune cell engagementin immune compromised cancer patients.

35. To be effective as a treatment method, it is desirable to achieve adegree of NK cell expansion that reaches an effective therapeutic dose.However, previous studies show that NK cell expansion was limited toseveral divisions and the cells achieved senescence and stoppedproliferating, coinciding with the observation of telomere shortening.While these methods allow for efficient in vitro NK cell expansion, theneed for live feeder cells makes the methodology difficult to transferto clinical settings that do not have large GMP facility and capability.Also, NK cells that are infused into the patient will likely stopdividing due to the lack of continued stimulation by the feeders.Furthermore, there is still a lack of information about the ability ofin vitro cultured NK cells to function as intended when re-infused intoa patient. Currently IL-2 administration is the only FDA approved methodof expansion of NK cells in vivo. However, the intensive conditioningregimen required for lymphodepletion and the high doses of IL-2 used inthis study resulted in significant toxicity and prolongedhospitalization, and in many cases, low in vivo expansion on NK cells.Moreover, systemic administration of IL-2 leads to proliferation ofregulatory T cells that suppress the numbers and function of NK cells,thereby limiting their persistence and efficiency in the patient. Thus,alternative approaches for in vivo or ex vivo expansion of NK cells areneeded. IL-15 is currently being tested in a Phase I clinical trial asan alternative approach to IL-2 administration but based on preclinicalfindings it is still expected to have significant toxicity ifadministered systematically. Thus, both methods carry significanttoxicities to patients and also induce proliferation of T-cellsincluding regulatory T-cells leading to short persistence (on averageless than 21 days) of NK cells.

36. As noted above, the efficacy of NK cell immunotherapy is dependenton the dose of NK cells administered to the patient or reached afterinfusion through in vivo expansion. Currently available techniques arelimited by their inability to achieve the level of NK cell expansionrequired to achieve a therapeutic effect in a patient. The lack of asimpler clinical expansion protocol is a major barrier to the progressand wide dissemination of NK cell-based immunotherapy. Current ex vivoexpansion protocols use a combination of high dose cytokines withactivating ligands expressed on leukemia-derived feeder/stimulator celllines, posing a significant disadvantages for transfer to clinicalsettings in most centers and are not amenable for direct in vivoexpansion. The use of particle technology, including exosomes, describedherein eliminates the need for stimulator cells, thus simplifying themethodology and allowing direct and selective in vivo expansion.Accordingly, and in one aspect, disclosed herein are methods forincreasing the number of memory NK cells comprising a) preactivating NKcells by contacting at least one NK cell with at least one or morestimulatory cytokines; and b) expanding the preactivated NK cells ofstep a) by contacting said cells with one or more vesicles comprising anNK cell effector agent.

37. The disclosed methods accomplish preactivation of NK cells bycontacting at least on NK cell with at least one or more stimulatorycytokines (for example IL-12, IL-15, and/or IL-18). Thus, in one aspect,disclosed herein are methods of increasing the number of memory NK cellscomprising preactivating NK cells by contacting one or more NK cellswith one or more stimulatory cytokines is selected from the groupcomprising IL-12, IL-15, and/or IL-18, or any combination thereof,including contacting one or more NK cells with 2 or 3 stimulatorycytokines. For example, specifically disclosed herein are methodswherein the preactivation step comprises contact NK cells with IL-12;11-15, IL-18, IL-12 and IL-15; IL-12 and IL-18; IL-15 and IL-18; orIL-12, IL-15, and IL-18. In one aspect, the disclosed methods ofincreasing the number of NK cells can further comprise contacting the NKcell with one or more cytokines selected from the group consisting of4-1BBL, IL-2, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155,CD112, CCR7, DAP12, and/or DAP10.

38. It is understood and herein contemplated that the duration ofpreactivation (i.e., the duration of contact between the NK cells andthe stimulatory cytokines (e.g., IL-12, IL-15, and/or IL-18) can be forany length of time necessary to achieve the desired preactivation of NKcells. For example, the contact can be as little as 1 minute or as muchas 7 days (for example, culturing the NK cells in the presence of IL-12,IL-15, and/or IL-18 for 7 days). In one aspect, disclosed herein aremethods of increasing the number of memory NK cells comprisingpreactivating NK cells by contacting one or more NK cells with IL-12,IL-15, and/or IL-18 for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 36, or 48 hours. It is understood and hereincontemplated that the half-life of a cytokine in culture may be lessthan the desired contact time. Accordingly, disclosed herein are methodswherein one or more NK cells are contacted with IL-12, IL-15, and/orIL-18 every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours within acontact period (for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 in a 24 hour contact period).

39. Through the use of plasma membrane (PM) particles, exosomes (EX), orfeeder cells (FC) comprising one or more NK cell effector agents (i.e.,stimulatory peptides, cytokines, and/or adhesion molecules) to contactand activate and/or expand NK cells many hurdles associated withcytokine toxicity are overcome. Examples of NK cell activating agentsand stimulatory peptides include, but are not limited to, 41BBL, IL-2,IL-12, IL-21, IL-18, MICA, LFA-1, 2B4, BCM/SLAMF2, CCR7 and/or otherhoming receptors. Examples of cytokines include, but are not limited to,IL-2, IL-12, IL-21, and IL-18. Examples of adhesion molecules include,but are not limited to LFA-1, MICA, BCM/SLAMF2. For example, a plasmamembrane (PM) particle, Feeder cells (FC), or exosomes (EX) preparedfrom feeder cells expressing membrane bound IL-21 (FC21 cells, PM21particles, and EX21 exosomes, respectively). The membrane bound IL-21expressing FC21 cells, PM21 particles, and EX21 exosomes can furthercomprise additional one or more activating agents, stimulatory peptides,cytokines, and/or adhesion molecules including, but not limited to41BBL, IL-2, IL-12, IL-15, IL-18, MICA, LFA-1, 2B4, BCM/SLAMF2, CCR7(for example, PM21 particle, EX21 exosome, or FC cell expressing 41BBLand membrane bound interleukin-21). Accordingly, in one aspect,disclosed herein are method for increasing the number of memory NK cellscomprising a) preactivating NK cells by contacting at least one NK cellwith at least one or more stimulatory cytokines; and b) expanding thepreactivated NK cells of step a) by contacting said cells with at leastone vesicle comprising an NK cell effector agent; wherein the NK celleffector agent comprising vesicle is any combination of one or more ofPM21 particle, EX21 particle, and/or FC21 feeder cells. For example,disclosed herein are methods of increasing memory NK cell numberscomprising, amongst other steps, expanding the preactivated NK cells ofstep a) by contacting said cells with at least one vesicle comprising anNK cell effector agent wherein the NK cell effector agent comprisingvesicle comprises PM21 particles; EX21 exosomes; FC21 feeder cells; PM21particles and EX21 exosomes; PM21 particles and FC21 feeder cells; EX21exosomes and FC21 feeder cells; or PM21 particles, EX21 exosomes, andFC21 feeder cells.

40. In some aspects, effector agents of the PM21 particles, EX21exosomes, or FC21 feeder cells comprise one or more stimulatory peptidescoupled to a membrane-inserting peptide (for example, Fc, GPI,trans-membrane T-cell receptor, or pHLIP). A membrane-inserting peptidemay be a molecule that promotes insertion into a membrane.Membrane-inserting peptides may comprise segments of CD4 or an IgG withaffinity for a lipid bilayer. In addition, alternativemembrane-inserting peptides may comprise human Fc, GPI, trans-membraneT-cell receptor, or pHLIP. The membrane self-inserting peptide may beany peptide known to insert into a cell membrane. Depending on the useof the membrane self-inserting peptide conjugate, certain membraneself-inserting peptides can be better choices than others. One of skillin the art would understand what membrane self-inserting peptide isideal under different circumstances. For example, for in vivo use, pHLIPmembrane self-inserting peptide may be suitable. pHLIP membraneself-inserting peptides insert into the membrane only under conditionsof low pH. Therefore, pHLIP conjugates will not insert into cellmembranes under normal physiological conditions. However, upon injectioninto a tumor environment, the pHLIP conjugate can insert into the cellmembrane of tumor cells because the tumor environment is more acidicthan normal physiological conditions. This insertion into the tumorenvironment allows for activation of NK cells in the area of the tumor.Using pHLIP thus prevents unwanted insertion into random cell membranes.

41. Membrane-inserting peptides may be coupled to one or morestimulatory peptides in a variety of ways and techniques for couplingpeptides are well known in the art. A membrane-inserting peptide coupledto a stimulatory peptide can also be referred to as a membrane-insertingpeptide conjugate. In some aspects, the one or more stimulatory peptidescoupled to a membrane-inserting peptide may comprise a fusion proteinencoded by recombinant DNA and such fusion-proteins may be produced inbacterial cells. In certain embodiments, fusion proteins may consist ofone or more stimulatory peptides conjugated or coupled to a lipophilicmolecule such as a hydrophobic peptide, GPI, or human Fc for anchoringinto liposomes or cellular membranes. cDNA vectors for these fusionproteins may be ligated into an expression plasmid, which allowsexpression in bacterial (E. coli), insect, or mammalian cells. Incertain embodiments, cDNA vectors may be FLAG- or HIS-tagged. Bacterialcells may be transfected using standard CaCl transfection methods, suchas that described in Sambrook et al., Molecular Cloning: A LaboratoryManual.2nd ed. Cold Spring Harbor Laboratory Press (1989). Bacterialcells may also be cultured in LB media and cells can be harvested andlysed using a French Press. Proteins of interest can be purified fromlysates by affinity chromatography. Palmitate-conjugated protein A andpurified Fc fusion proteins can be conjugated as described in theliterature by mixing 1:2 (w/w) at 4 degrees C. The conjugates may thenbe directly injected intratumorally or may be incorporated intoliposomes.

42. Types of coupling and methods for coupling are known to thoseskilled in the art. As used herein, term “couple” refers to the membraneself-inserting peptide being conjugated, connected, or otherwise linkedto another molecular entity such as a peptide or protein. For example,membrane-inserting peptides coupled to stimulatory peptides can befusion proteins wherein the membrane-inserting peptide is coupled toanother protein via a disulfide bond. Coupling or conjugating may meanthat there is a chemical linkage between the membrane self-insertingpeptide and the NK cell effector agent.

43. In some aspects, one or more stimulatory peptides may be coupled tomembrane self-inserting peptides or GPI anchors for in situself-assembly. For example, 41-BBL and IL-21 may be coupled to a pHLIPpeptide which inserts itself into cellular membranes under acidicconditions, thereby allowing the anchoring of the stimulatory ligandsinto cells in the proximity of tumor. The stimulatory peptides 41BBL,IL-2, IL-12, IL-21, BCM/SLAMF2, CCR7 and/or other homing receptors maybe produced in bacterial cells or purchased from commercially availablesources and cDNA vectors for these proteins may optionally be ligatedinto pTriEX expression plasmid which allows expression in bacterial (E.coli), insect, or mammalian cells. The cDNA vector may code forexpression of FLAG- or HIS-tag. Bacterial cells can be transfected usingstandard CaCl transfection methods and may be cultured on LB media.Cells can be harvested and lysed using a French press and proteins ofinterest may then be purified from lysates by affinity chromatography.

44. In some embodiments, pHLIP may be prepared by solid-phase peptidesynthesis using 9-fluorenylmethyloxycarbonyl chemistry and the productmay be purified on a C18 column by reverse-phase chromatography. pHLIPmay then be conjugated to stimulatory human protein ligands byincubating with a crosslinker, such as benzophenone-4-iodoacetamide.After several washes, the conjugated pHLIP protein may be resuspended inmedia (saline, for example) and injected intratumorally orintravenously. Based on evidence from prior literature and presented inexperimental results, interaction of NK cells with stimulatory ligandssuch as IL-21 and 41-BBL on the surface of such modified tumor cells maystimulate in situ NK cell expansion and trigger their cytotoxic responsetoward a tumor. This type of stimulatory approach can be used fortreatments of solid tumors such as ovarian cancer where NK stimulatoryligands that insert in situ into tumor cells under acidic pH can beinjected into intraperitoneal space of patients with low dose IL-2 aloneor together with NK cells. There is strong evidence that cytotoxiclymphocytes that express high levels of FCγIII R (CD16) such as NK cellsare crucial for the efficacy of cancer therapy with therapeuticantibodies. Thus, this approach can also be used in combination withtherapeutic antibodies.

45. It is understood and herein contemplated that the duration ofcontact between the preactivated NK cells (i.e., NK cells contacted witha cytokine such as IL-12, IL-15, and/pr IL-18) and the NK cell effectoragent comprising vesicle (i.e., PM21 particles, EX21 exosomes, and/orFC21 feeder cells) can be for any length of time necessary to achievethe desired expansion of memory NK cells. For example, the contact canbe as little as 1 minute or as much as 60 days (for example, culturingthe NK cells in the presence of PM21 particles, EX21 exosomes, and/orFC21 feeder cells for 7 days). In one aspect, the contact between thepreactivated NK cells and the NK cell effector agent comprising vesiclecan be between about 6 days and about 60 day, more preferably thecontact can be between about 6 days and about 40 days. Also disclosedherein are methods of increasing the number of memory NK cellscomprising contacting the preactivated NK cells with PM21 particles,EX21 exosomes, and/or FC21 feeder cells for 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, or 72 days. It is understood and herein contemplatedthat in some instances, multiple contact of the preactivated NK cellswith PM21 particles, EX21 exosomes, and/or FC21 feeder cells may bedesired and can be employed. For example, the preactivated NK cells canbe contacted with the PM21 particles, EX21 exosomes, and/or FC21 feedercells once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 hrs, 2,3, 4, 5, 6, 7, 8, 9, 0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 days. Accordingly, in one aspect, disclosed herein are are methods ofincreasing the number of memory NK cells comprising contacting thepreactivated NK cells with PM21 particles, EX21 exosomes, and/or FC21feeder cells more than one time, wherein the contact occurs every 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 hrs, 2, 3, 4, 5, 6, 7, 8, 9, 0,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

46. In one aspect, the plasma membrane particles, feeder cells, orexosomes can be purified from feeder cells that stimulate NK cell. NKcell stimulating feeder cells for use in the claimed invention, for usein making the plasma membrane particles or exosomes, disclosed hereincan be either irradiated autologous or allogeneic peripheral bloodmononuclear cells (PBMCs) or nonirradiated autologous PBMCs, RPMI8866,HFWT, K562, K562 cells transfected with membrane bound IL-15 and 41BBL,K562 cells transfected with membrane bound IL-21 and 41BBL, or EBV-LCL.In some aspects, the NK cell feeder cells can be K562 cells transfectedwith membrane bound IL-21 and 41BBL or K562 cells transfected withmembrane bound IL-15 and 41BBL.

47. It is understood and herein contemplated that the use of aparticular stimulatory cytokine in the preactivation step has no bearingon the NK cell effector agent comprising vesicle used for expanding thepreactivated NK cells. Thus, any combination of cytokines and vesiclescan be used in the disclosed methods. For example, specificallydisclosed herein are methods for increasing the number of memory NKcells comprising a) preactivating NK cells by contacting at least one NKcell with at least one or more stimulatory cytokines; and b) expandingthe preactivated NK cells of step a) by contacting said cells with oneor more vesicles comprising an NK cell effector agent (for example,contacting with PM21 particles, EX21 exosomes, or FC21 feeder cells);wherein the one or more stimulatory cytokines is IL-12, IL-15, and/orIL-18 and the one or more vesicles are PM21 particles, EX21 exosomes,and/or FC21 feeder cells. For example, disclosed herein are methodscomprising IL-12 and PM21 particles; IL-15 and PM21 particles; IL-18 andPM21 particles; IL-12 and EX21 exosomes, IL-15 and EX21 exosomes; IL-18and EX21 exosomes; IL-12 and FC21 feeder cells; IL-15 and FC21 feedercells; IL-18 and FC21 feeder cells; IL-12, IL15, and PM21 particles;IL-12, IL-18, and PM21 particles; IL-15, IL-18, and PM21 particles;IL-12, IL-15, IL-18, and PM21 particles; IL-12, IL15, and EX21 exosomes;IL-12, IL-18, and EX21 exosomes; IL-15, IL-18, and EX21 exosomes; IL-12,IL-15, IL-18, and EX21 exosomes; IL-12, IL15, and FC21 feeder cells;IL-12, IL-18, and FC21 feeder cells; IL-15, IL-18, and FC21 feedercells; IL-12, IL-15, IL-18, and FC21 feeder cells; IL-12, EX21 exosomes,and PM21 particles; IL-15, EX21 exosomes, and PM21 particles; IL-18,EX21 exosomes, and PM21 particles; IL-12, FC21 feeder cells, and PM21particles; IL-15, FC21 feeder cells, and PM21 particles; IL-18, FC21feeder cells, and PM21 particles; IL-12, FC21 feeder cells, and EX21exosomes; IL-15, FC21 feeder cells, and EX21 exosomes; IL-18, FC21feeder cells, and EX21 exosomes; IL-12, FC21 feeder cells, PM21particles, and EX21 exosomes; IL-15, FC21 feeder cells, PM21 particles,and EX21 exosomes; IL-18, FC21 feeder cells, PM21 particles, and EX21exosomes; IL-12, IL15, EX21 exosomes, and PM21 particles; IL-12, IL-18,EX21 exosomes, and PM21 particles; IL-15, IL-18, EX21 exosomes, and PM21particles; IL-12, IL-15, IL-18, EX21 exosomes, and PM21 particles;IL-12, IL15, FC21 feeder cells, and PM21 particles; IL-12, IL-18, FC21feeder cells, and PM21 particles; IL-15, IL-18, FC21 feeder cells, andPM21 particles; IL-12, IL-15, IL-18, FC21 feeder cells, and PM21particles; IL-12, IL15, EX21 exosomes, and FC21 feeder cells; IL-12,IL-18, EX21 exosomes, and FC21 feeder cells; IL-15, IL-18, EX21exosomes, and FC21 feeder cells; IL-12, IL-15, IL-18, EX21 exosomes, andFC21 feeder cells; IL-12, EX21 exosomes, FC21 feeder cells, and PM21particles; IL-15, EX21 exosomes, FC21 feeder cells, and PM21 particles;IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-12,IL15, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-12,IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-15,IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; and IL-12,IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles.

48. It is understood and herein contemplated that each step of thedisclosed methods of increasing memory NK cell numbers can occur invitro, in vivo, or ex vivo. That is, the peactivation of NK cells withone or more cytokines can occur in vitro, in vivo, or ex vivo.Similarly, contact of the preactivated NK cells with PM21 particles,EX21 exosomes, and/or FC21 feeder cells can occur in vitro, in vivo, orex vivo. Also, the resting of the memory NK cells can occur PM21particles, EX21 exosomes, and/or FC21 feeder cells. Additionally it isunderstood that whether a step occurs in vitro, ex vivo, or in vivo isentirely independent of the step preceding or subsequent to a particularstep. For example, the preactivation can occur in vitro or ex vivo whilethe contacting of the preactivated NK cells with PM21 particles, EX21exosomes, and/or FC21 feeder cells occurs in vivo. Alternatively, thepreactivation; contact with PM21 particles, EX21 exosomes, and/or FC21feeder cells; and resting can occur all in vitro or ex vivo.Additionally, the preactivated NK cells can be contacted to PM21particles, EX21 exosomes, and/or FC21 feeder cells in an allogeneictransplant procedure, a haploidentical transplant procedure or an invivo immunotherapy procedure. In some aspects, the use of NK-stimulatingexosomes in allogeneic transplants, haploidentical transplants or invivo immunotherapy does not cause graft-versus-host-disease (GVHD).

49. In one aspect, it is contemplated herein that the disclosed methodscan be used with NK cells obtained from any donor source including NKcells obtained from an unselected population of peripheral bloodmononuclear cells. In some instances the donor source for the NK cellsbeing used in the disclosed kits for increasing the number of memory NKcells can also be the recipient for the NK cells. Accordingly, the NKcells can be from an autologous source. In other instances the donorsource for the NK cells can be a haploidentical or allogeneic donorsource. In one aspect, the source of the NK cells can be any sourcewhere NK cell progenitor cells may be found including, but not limitedto, cord blood or stem cell source such as induced pluripotent stemcells

It is understood that a period of rest can benefit the resulting memoryNK cells by allowing rapid and vigorous production of IFN-γ when it isre-introduced to a tumor or restimualted by cytokines, at the tumorsite. The value of having rapid and vigorous IFN-γ production at thetumor site upon cytokine restimulation or following being introduced toa tumor is three fold: 1) the NK cells don't run out of bullets (bothIFN-γ and granzyme); 2) the NK cells last longer in circulation; and 3)the anti-tumor effect with the increased IFN-g is targeted and lessgeneral systemic inflammation. Accordingly, and in one aspect, disclosedherein are methods of increasing the number of memory NK cells furthercomprising resting the memory NK cells following the expansion step b)for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or 21 days.

In one aspect, it is appreciated that washing the cells between or aftersteps can more precisely control exposure to cytokines and/or NK celleffector agent particles or prevent contamination of subsequent steps ofthe disclosed method or usages of the memory NK cells. Washing can occurin any manner acceptable in the art including, but not limited to,cycles of centrifugation and resuspension in an acceptable wash followedby a final resuspension in media, simple pouring off of culture mediafollowed by one or more rinses with an acceptable media; said washingbeing conducted at culture temperature, room temperature, or a cold wash(e.g, over ice or in a refrigerated centrifuge). The washes can beaccomplished phosphate buffered saline (PBS) or with any appropriatemedia for culturing NK cells (for example, CELLGRO® GMP media, RoswellPark Memorial Institute (RPMI) institute, Minimal Essential Media (MEM),Eagle's MEM (EMEM), X-VIVO 20™, etc.) with or without serum (e.g., fetalbovine serum). Thus, in one aspect, disclosed herein are methods ofincreasing the number of NK cells comprising a) preactivating NK cellsby contacting at least one NK cell with at least one or more stimulatorycytokines; and b) expanding the preactivated NK cells of step a) bycontacting said cells with PM21 particles, EX21 exosomes, or FC21 feedercells; further comprising washing the NK cells after preactivation withIL-12, IL-15, and IL-18 and/or washing the NK cells after expansion withone or more vesicle comprising an NK cell effector agent (i.e., PM21particles, EX21 exosomes, and/or FC21 feeder cells).

The disclosed methods provide a surprising increase in the number ofmemory NK cells significantly beyond numbers achieved by alternativemethods. As shown in FIG. 1, preactication with stimulatory cytokinesfollowed by expansion of the preactivated cells through contact withPM21 particles expanded NK cell number over 10,000 fold in 15 days. Thisis a 8-fold increase over expansion with K562-mb21 feeder cells or and100-fold increase over expansion with PM21 particles alone.

C. Compositions

50. Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular cytokine (for example IL-12, IL-15, or IL-18),PM21 particle, EX21 exosome, or FC21 feeder cell is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules including the cytokine (for example IL-12, IL-15, or IL-18),PM21 particle, EX21 exosome, or FC21 feeder cell are discussed,specifically contemplated is each and every combination and permutationof the cytokine (for example IL-12, IL-15, or IL-18), PM21 particle,EX21 exosome, or FC21 feeder cell and the modifications that arepossible unless specifically indicated to the contrary. Thus, if a classof molecules A, B, and C are disclosed as well as a class of moleculesD, E, and F and an example of a combination molecule, A-D is disclosed,then even if each is not individually recited each is individually andcollectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F,C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupof A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

51. The disclosed methods increasing the number of memory NK cellsutilize one or more cytokines (for example, IL-12, IL-15, and/or IL-18)in combination with a vesicle comprising an NK cell effector agent, suchas, for example, PM21 particles, FC21 feeder cells, and/or EX21exosomes. It is understood and herein contemplated that it would beadvantageous to provide the components utilized in the disclosed methodsin a package that would readily allow a person to perform the disclosedmethods.

Thus, in one aspect, disclosed herein are kits for increasing the numberof memory NK cells comprising one or more cytokines (for example, IL-12,IL-15 and/or IL-18) and one or more vesicle comprising an NK celleffector agent. In one aspect, the vesicle can be PM21 particles, EX21exosomes, and/or FC21 feeder cells. For example, the disclosed kits cancomprise IL-12 and PM21 particles; IL-15 and PM21 particles; IL-18 andPM21 particles; IL-12 and EX21 exosomes, IL-15 and EX21 exosomes; IL-18and EX21 exosomes; IL-12 and FC21 feeder cells; IL-15 and FC21 feedercells; IL-18 and FC21 feeder cells; IL-12, IL15, and PM21 particles;IL-12, IL-18, and PM21 particles; IL-15, IL-18, and PM21 particles;IL-12, IL-15, IL-18, and PM21 particles; IL-12, IL15, and EX21 exosomes;IL-12, IL-18, and EX21 exosomes; IL-15, IL-18, and EX21 exosomes; IL-12,IL-15, IL-18, and EX21 exosomes; IL-12, IL15, and FC21 feeder cells;IL-12, IL-18, and FC21 feeder cells; IL-15, IL-18, and FC21 feedercells; IL-12, IL-15, IL-18, and FC21 feeder cells; IL-12, EX21 exosomes,and PM21 particles; IL-15, EX21 exosomes, and PM21 particles; IL-18,EX21 exosomes, and PM21 particles; IL-12, FC21 feeder cells, and PM21particles; IL-15, FC21 feeder cells, and PM21 particles; IL-18, FC21feeder cells, and PM21 particles; IL-12, FC21 feeder cells, and EX21exosomes; IL-15, FC21 feeder cells, and EX21 exosomes; IL-18, FC21feeder cells, and EX21 exosomes; IL-12, FC21 feeder cells, PM21particles, and EX21 exosomes; IL-15, FC21 feeder cells, PM21 particles,and EX21 exosomes; IL-18, FC21 feeder cells, PM21 particles, and EX21exosomes; IL-12, IL15, EX21 exosomes, and PM21 particles; IL-12, IL-18,EX21 exosomes, and PM21 particles; IL-15, IL-18, EX21 exosomes, and PM21particles; IL-12, IL-15, IL-18, EX21 exosomes, and PM21 particles;IL-12, IL15, FC21 feeder cells, and PM21 particles; IL-12, IL-18, FC21feeder cells, and PM21 particles; IL-15, IL-18, FC21 feeder cells, andPM21 particles; IL-12, IL-15, IL-18, FC21 feeder cells, and PM21particles; IL-12, IL15, EX21 exosomes, and FC21 feeder cells; IL-12,IL-18, EX21 exosomes, and FC21 feeder cells; IL-15, IL-18, EX21exosomes, and FC21 feeder cells; IL-12, IL-15, IL-18, EX21 exosomes, andFC21 feeder cells; IL-12, EX21 exosomes, FC21 feeder cells, and PM21particles; IL-15, EX21 exosomes, FC21 feeder cells, and PM21 particles;IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-12,IL15, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-12,IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-15,IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; or IL-12,IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles.

52. It is understood and herein contemplated that the NK cell effectoragents comprised in the vesicles (e.g., PM21 particles, EX21 exosomes,and/or FC21 feeder cells) can be selected from the group of NK celleffector agents consisting of 4-1BBL, IL-2, IL-21, MICA/B, ULBP2,ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

53. It is understood and herein contemplated that the disclosed kits cancomprise cytokines in addition to IL-12, IL-15, and/or IL-18.Accordingly, in one aspect are kits for increasing the number of NKcells further comprising 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA/B,ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

54. In one aspect, it is contemplated herein that the disclosed kits canbe used with NK cells obtained from a donor source including NK cellsobtained from an unselected population of peripheral blood mononuclearcells. In some instances the donor source for the NK cells being used inthe disclosed kits for increasing the number of memory NK cells can alsobe the recipient for the NK cells. Accordingly, the NK cells can be froman autologous source. In other instances the donor source for the NKcells can be a haploidentical or allogeneic donor source.

55. It is further contemplated herein that there are instances where itwould be beneficial to provide NK cells in the kit. Accordingly in oneaspect, disclosed herein are kits for increasing the number of memory NKcells further comprising NK cells or an NK cell line.

1-20. (canceled)
 21. A method for increasing the number of memory NKcells comprising a) preactivating NK cells by contacting at least one NKcell with at least one stimulatory cytokine; and b) expanding thepreactivated NK cells of step a), by contacting the preactivated NKcells with plasma membrane (PM) particles with surface bound interleukin(IL)-21 (PM21) particles, exosomes (EX) with surface bound IL-21 (EX21)exosomes, or feeder cells (FC) with surface bound IL-21 (FC21) feedercells, or any combination thereof.
 22. The method of claim 21, furthercomprising maintaining the contact in (b) for at least about 15 days,wherein a 10,000 fold increase in NK cell number is achieved.
 23. Themethod of claim 21, further comprising a washing step after thepreactivation in step (a) and/or after the expansion in step (b). 24.The method of claim 21, wherein the NK cells are obtained from anunselected population of peripheral blood mononuclear cells, cord bloodor a stem cell population.
 25. The method of claim 21, whereinpreactivating the NK cells comprises contacting the NK cells with atleast one stimulatory cytokine selected from the group consisting ofInterleukin-12 (IL-12), Interleukin-15 (IL-15), and Interleukin-18(IL-18).
 26. The method of claim 21, wherein preactivating the NK cellscomprises contacting the NK cells with any combination of twostimulatory cytokines selected from the group consisting of IL-12,IL-15, and IL-18.
 27. The method of claim 21, wherein preactivating theNK cells comprises contacting the NK cells with IL-12, IL-15, and IL-18.28. The method of claim 24, further comprising contacting the NK cellswith an one or more NK cell effector agents selected from the groupconsisting of 4-1BB ligand (4-1BBL), Interleukin-2 (IL-2), Majorhistocompatibility complex class I-related chain AB (MICAS), UL16Binding Protein 2 (ULBP2), Intracellular adhesion molecule 1(ICAM-1),2B4, /signaling lymphocyte activation molecule (SLAM) family member 2(F2) (BCM1/SLAMF2), cluster of differentiation 155 CD155), cluster ofdifferentiation 112 (CD112), C-C chemokine receptor type 7 (CCR7), DnaXactivation protein of 12 kDa (DAP12), and DnaX activation protein of 10kDa (DAP10), and any combination thereof.
 29. The method of claim 21,wherein preactivating the NK cells comprises contacting the NK cellswith the at least one stimulatory cytokine in vitro, in vivo, or exvivo.
 30. The method of claim 21, wherein preactivating the NK cellscomprises contacting the NK cells with at least one stimulatory cytokinefor a period of about 6 to about 24 hours.
 31. The method of claim 21,wherein the PM21 particles, EX21 exosomes, or FC21 feeder cells compriseone or more stimulatory peptides coupled to a membrane-insertingpeptide.
 32. The method of claim 31, wherein the stimulatory peptidecoupled to a membrane-inserting peptide comprises a fused peptide thatis capable of membrane insertion with affinity for a lipid bilayer,wherein the fused peptide comprises a segment of IG4, CD4, or acombination thereof.
 33. The method of claim 31, wherein the one or morestimulatory peptides coupled to a membrane-inserting peptide is a fusionprotein encoded by recombinant DNA.
 34. The method of claim 31, whereinthe membrane-inserting peptide comprises human Fc, GPI, trans-membraneT-cell receptor, or pHLIP.
 35. The method of claim 31, wherein the oneor more stimulatory peptides are selected from the group consisting of4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA/B, ULBP2, ICAM-1,2B4,BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.
 36. The method ofclaim 21, wherein the NK cells are contacted with the PM21 particles,EX21 exosomes, or FC21 feeder cells in vitro, in vivo, or ex vivo. 37.The method of claim 21, wherein the NK cells of step (a) are contactedwith PM21 particles, EX21 exosomes, or FC21 feeder cells for a period of7 days to 60 days.
 38. The method of claim 21, further comprisingresting the expanded memory NK cells following step (b).
 39. The methodof claim 38, wherein the cells are memory NK cells are rested for atleast 1, 2, 3, 4 or 5 days.
 40. The method of claim 38, wherein thememory NK cells are rested for no more than 5 days.
 41. The method ofclaim 21, wherein the PM21 particles, EX21 exosomes, or FC21 feedercells comprise 4-1BBL and IL-21.
 42. The method of claim 41, wherein the4-1BBL and IL-21 are coupled to a membrane-inserting peptide.
 43. Anactivated NK cell composition comprising NK cells following contact withat least one stimulatory cytokine selected from the group consisting ofIL-12, IL-15, and IL-18 for at least about 6 hours.
 44. The cellcomposition of claim 43, in contact with PM21 particles, EX21 exosomes,FC21 feeder cells, or any combination thereof.
 45. The composition ofclaim 44, further comprising an additional one or more cytokinesselected from the group consisting of 4-1BBL, IL-2, IL-21, MICA/B,ULBP2, ICAM-1, 2B4BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, DAP10, and anycombination thereof.
 46. The composition of claim 44, in vitro or exvivo.
 47. The composition of claim 44, wherein the PM21 particles, EX21exosomes, or FC21 feeder cells comprise one or more stimulatory peptidescoupled to a membrane-inserting peptide.
 48. The composition of claim47, wherein each of the one or more stimulatory peptides coupled to amembrane-inserting peptide comprises a fused peptide that is capable ofmembrane insertion with affinity for a lipid bilayer, wherein the fusedpeptide comprises a segment of IG4, CD4, or a combination thereof. 49.The composition of claim 47, wherein the one or more stimulatorycytokines coupled to a membrane-inserting peptide is a fusion proteinencoded by recombinant DNA.
 50. The composition of claim 47, wherein themembrane-inserting peptide comprises human Fc, GPI, trans-membraneT-cell receptor, or pHLIP.
 51. The composition of claim 47, maintainedin contact with the PM21 particles, EX21 exosomes, FC21 feeder cells orcombination thereof for a period of 7 days to 60 days.
 52. Thecomposition of claim 47, wherein the PM21 particles, EX21 exosomes, orFC21 feeder cells comprise IL-21 and further comprise 4-1BBL.
 53. Themethod of claim 51, wherein the 4-1BBL and IL-21 are coupled to amembrane-inserting peptide.
 54. A kit for increasing the number ofmemory NK cells comprising amounts of one or more stimulatory cytokinesand amounts of one or more vesicles comprising IL-21, and optionallyamounts of one or more NK cell effector agents.
 55. The kit of claim 54,wherein the one or more stimulatory cytokines are selected from thegroup consisting of IL-12, IL-15, IL-18 and any combination thereof. 56.The kit of claim 54, wherein the one or more vesicles comprising IL-21are selected from a PM21 particle, an EX21 exosome, a FC21 feeder celland any combination thereof.
 57. The kit of claim 54, wherein the one ormore NK cell effector agents are selected from the group consisting of41BBL, IL-2, IL-15, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2,CD155, CD112, CCR7, DAP12, DAP10 and any combination thereof.
 58. Thekit of claim 54, further comprising an amount of NK cells or an NK cellline.